1. Field of the Invention
The present invention relates to novel microorganisms and to a novel biotechnological process for preparing or enriching N-acetylmannosamine using these microorganisms.
2. Background Art
N-Acetylmannosamine (hereinbelow referred to as NAM) is an important intermediate in the preparation of N-acetylneuraminic acid (hereinbelow referred to as NANA), which in turn is an important starting material for therapeutic agents (European Published Patent Application No. 0701623).
To date, a plurality of biotechnological process for preparing or enriching NAM are known. Spivak C. T. and Roseman S. (JACS, Volume 81, 1959, pp. 2403 to 2404) describe the enrichment of NAM using washed microorganisms of the species E. coli, which have been adapted such that they can grow using N-acetylglucosamine (hereinbelow referred to as NAG) as the sole carbon source. This process has the disadvantages that it is not feasible industrially, and that NAM is obtained in only moderate yield.
Kuboki et al., (Tetrahedron, Vol. 53, 1997, pp. 2387 to 2400), describes a process for enriching NAM using washed microorganisms of the species Rhodococcus rhodochrous, where NAM is enriched starting from a mixture comprising NAM and NAG in a ratio of 4.5:1 (82:18). In this process, too, NAM is enriched in an uneconomical, not industrially feasible, manner.
The object of the present invention is to provide a microorganism and a biotechnological process for preparing or enriching NAM, where the desired product is enriched rapidly in a simple manner. This object is achieved with microorganisms which are in accordance with the present invention obtainable by selecting microorganisms using N-Ac-Glc rapidly as sole carbon source for growth and are stable and can metabolize N-Ac-Glc rapidly in the presence of N-Ac-Man and with a process employing said microorganisms for preparing N-Ac-Man by the microorganism of the invention and the process of the invention.
The microorganisms according to the invention can be isolated from soil samples, mud or wastewater with the aid of customary microbiological techniques. According to the invention, the microorganisms are isolated in such a manner that they are cultivated in a customary manner in a medium comprising NAG as the sole carbon source, and with a suitable nitrogen source. From the culture obtained by cultivation, those are selected which are stable and have the property to metabolize NAG rapidly in the presence of NAM.
The invention includes biologically pure cultures of the microorganisms of the invention.
Advantageously, the microorganisms selected in such a manner metabolize NAG in a concentration of 60 g/l in less than 30 hours.
A suitable nitrogen source which can be used by the microorganisms is, for example, ammonium.
The selection medium used can be the mineral salt media customarily used by persons skilled in the art, such as, the medium described in Table 1, the mineral salt medium according to Kulla et al., Arch. Microbiol. (1983), 135, 1 to 7, or buffers of low molarity comprising mineral salts/trace elements. Preference is given to using the medium described in Table 1 of Kulla et al, ibid. The buffer of low molarity used can, for example, be a phosphate buffer of low molarity.
The selection is generally carried out at a temperature of from 15xc2x0 to 60xc2x0 C., advantageously from 25xc2x0 to 45xc2x0 C., and at a pH between pH 4 and pH 9, advantageously between pH 6 and pH 8.
Preferred microorganisms are microorganisms of the genus Klebsiella, advantageously of the species Klebsiella pneumoniae having the designation KHA (DSM 12702) and KHA1 (DSM 12703) or their xe2x80x9cfunctionally equivalent variantsxe2x80x9d. The microorganisms having the designation DSM 12702 and DSM 12703 were deposited with the Deutsche Sammiung von Mikroorganismen und Zellkultur GmbH, Mascheroderweg 1b, D-38124 Brunswick, in accordance with the Budapest Treaty, on Feb. 23, 1999.
xe2x80x9cFunctionally equivalent variantsxe2x80x9d are to be understood as microorganisms which essentially have the same properties and functions as the original microorganisms. Such variants can be formed arbitrarily, for example, by UV irradiation or other mutagenesis techniques known to a person skilled in the art.
The process of according to the invention for preparing or enriching NAM is carried out such that, starting from an NAG/NAM mixture, NAG is metabolized by fermentation using the microorganisms according to the invention, NAM being enriched.
Advantageously, NAG is employed as a mixture comprising NAG and NAM in a ratio by weight of 1:1. This mixing ratio is preferably, according to R. Kuhn and G. Barschang (Liebigs Ann., 659, 1962, pp. 156 to 163), obtained by epimerizing the NAG, which was hydrolyzed from chitin, in a basic medium, resulting in a thermodynamical equilibrium of NAG to NAM of 4:1 parts by weight. This mixture is then enriched in accordance with the literature reference described above by selective removal of NAG, for example, by crystallization, so that a mixing ratio of NAM:NAG of 1:1 results.
In principle, the further enrichment according to the invention of NAM is possible using any NAG-metabolizing microorganisms which can be obtained by the selection method already described. In particular, the enrichment is carried out using the microorganisms of the genus Klebsiella, more preferably of the species Klebsiella pneumoniae. In a most preferred embodiment, the invention is carried out using strains of Klebsiella pneumoniae having the designation KHA (DSM 12702) or KHA1 (DSM 12703) or their xe2x80x9cfunctionally equivalent variantsxe2x80x9d.
The enrichment of NAM is advantageously carried out directly by fermentation of the selected microorganisms on the NAG/NAM mixture (i.e., with growing cells under aerobic conditions), without removing and washing the microorganisms beforehand, for example, by centrifugation.
Suitable for use as fermentation media are the same media as those described above used for the selection, using the NAG/NAM mixture instead of NAG as the carbon source.
Advantageously, the metabolization of NAG is carried out such that the concentration is below 20 percent by weight, preferably below 10 percent by weight. In particular, the appropriate NAG/NAM mixture is added once, batch-wise (in a plurality of portions) or continuously. The pH of the medium can be in the range of from 5 to 9, preferably from 6 to 8. The metabolization is advantageously carried out at a temperature of from 20xc2x0 to 50xc2x0 C., preferably from 25 to 40xc2x0 C.
After customary metabolization of less than 30 hours, the NAM enriched in this manner can be isolated by customary work-up methods, such as, by electrodialysis, filtration techniques and crystallization